One of the histological findings necessary for making a diagnosis of Alzheimer's disease (AD) is the cytoskeletal pathology termed the neurofibrillary tangle (NFT). The microtubule associated protein, tau, is the primary component of the NFT. Attempts to define a mechanism of NFT formation often focus on site specific phosphorylations of tau protein. These have been described in postmortem tissue primarily in developing and AD affected brains and are thought to be a preliminary event in the formation of the tangle. Study of the developmental regulation of tau's Alzheimer epitope phosphorylations may help explain their persistence or recurrence during the pathogensis of AD. To begin examining the developmental tau phosphorylations, we have used rat embryonic hippocampal cultures. Using phosphorylation dependent anti-tau antibodies, a temporal phosphorylation of two Alzheimer epitopes has been seen during neuronal maturation in vitro. It should be possible to characterize potential kinase regulatory systems for these discreet phosphorylation events. Once these kinase actions have been identified, it will be reasonable to expect similar events to be recapitulated or maintained within neurons bearing hyperphosphorylated tau and NFTs in Alzheimer's Disease brain. Hypothesis I: Neurons express independent temporal phosphorylations of tau's AD epitopes during development in vitro. This can be addressed in the following manner: Aim 1: Tau's intracellular localization and temporal phosphorylation on selected Alzheimer epitopes in vitro will be defined using immunohistochemistry. This will be supported by Western blot analysis. Hypothesis II: It if further hypothesized that tau's specific phosphorylation state in developing neurons in vitro will correspond to expression and/or activation of specific kinases. This question will be answered by the following: Aim 2: The development expression of selected kinases will be compared to the temporal pattern of tau's site specific phosphorylations defined in Aim 1. This will be done using double-label immunofluorescent histochemistry. As additional evidence for the involvement of selected kinases in the regulation of tau phosphorylation, specific kinases will be pharmacologically inhibited in culture and tau phosphorylation state will be monitored for resulting changes via immunohistochemistry and Western blot analysis. Selected kinase activity assays will be necessary to determine the drug concentrations needed for maximal inhibition.